We will investigate the molecular mechanisms of metalloenzyme catalysis through the use of recombinant DNA methodology. We seek to provide the structure-function link in aspects of macromolecular recognition, the regio- and stereospecificity of carbon chain functionalization, the chemical mechanisms of oxidative catalysis, and the role of metal center ligands in determining the electronic and spectroscopic properties of heme and iron-sulfur centers. Specifically, we are interested in the mechanisms by which a macromolecule recognizes both its small molecule substrate as well as the ancillary proteins necessary to form metalloenzyme complexes of the precisely defined topology cytochrome P-450cam, cytochrome b5, cytochrome c, and myoglobin where in all cases the three dimensional x-ray structures are known to high resolution. Protein-protein recognition is studied by surface charge mutagenesis and high pressure spectroscopy. We are using site directed mutagenesis to examine the mechanisms of diatomic ligand discrimination that allows oxygen transport, storage, and respiration to occur under the normal physiological levels of carbon monoxide production which would otherwise poison heme proteins. The specificity, both in terms of regio- and stereoselectivity, of cytochrome P-450cam is also being examined as a question of molecular recognition, and our initial results have indicated the feasibility of complete re- engineering of an enzyme active site for de novo design of catalytic processing. A final major specific aim of our continuing work is to delineate the chemical mechanisms of catalysis as dictated by the specific requirement of individual amino acid side chains in the active site environment. For example, by alteration of metal center ligands (histidine, tyrosine, and cysteine) we have been able to generate new catalytic activities of metalloproteins. Provision of new active site acid-base functions can open the possibility for development of more efficient and novel catalysts. Mutagenesis of aromatic amino acids is being used to define path-dependent electron transfer reactions. In summary, GM33775 brings the powerful techniques of recombinant DNA technology to bear on the important problems in metalloenzyme mechanisms.